Screening process for atopic dermatitis

ABSTRACT

The present invention provides a process of screening patients for atopic dermatitis. The process includes the step of determining, in sera of the patient, the presence of antibodies against nuclear antigens such as transcription co-activator p75, wherein the presence of such antibodies indicates atopic dermatitis. The screening process can be used to detect atopic dermatitis in patients suffering from other conditions such as asthma or interstitial cystitis.

[0001] Funds used to support some of the studies reported herein wereprovided by the National Institutes of Health (NIR grants DK49413 andAR32063). The United States Government may, therefore, have certainrights in any invention disclosed herein.

TECHNICAL FIELD OF THE INVENTION

[0002] The field of this invention is diagnosis of atopic dermatitis.

BACKGROUND OF THE INVENTION

[0003] Atopic dermatitis (AD) is a chronic, relapsing, pruritic skindisorder that generally first appears in childhood and frequentlyprogresses to asthma and/or allergic rhinitis in the adult (Hanifin etal., Acta Dermatovener (Stockholm) Suppl 1980; 92:44-47; Leung et al.,Dermatology in General Medicine, 4th edition, 1993, pp. 1543-1564;Cooper, J Invest Dermatol 1994; 102:128-137). There are no reliablelaboratory markers for this condition but AD patients often haveelevated serum IgE levels, allergic reactivity to foods and to othercommon allergens such as pollens, molds, and insects. There have alsobeen reports of antinuclear antibodies (ANAs) in this condition(Taniguchi et al., Acta Derm Venereol (Stockh) Suppl 1992; 176:62-64;Tada et al., Dermatol 1994; 189:38-40). The prevalence of AD appears tobe on the rise, with 10-15% of the population being affected at sometime during childhood (Beltrani, J Allergy Clin Immunol 1999;104:587-598; Leung, J Allergy Clin Immunol 1999; 104: S99-S108). We nowreport the finding of an autoantibody-autoantigen system in 30% ofpatients with AD which is also shared to a lesser extent by patientswith asthma and interstitial cystitis (IC). IC is a urinary bladdercondition in which the classical pathology is characterized bypredominant mononuclear cell infiltration of the lamina propria withlymphocytes, plasma cells and mast cells (Gillenwater, et al., J Urol1988; 140:203-206).

BRIEF SUMMARY OF THE INVENTION

[0004] The present invention provides a process of screening patientsfor atopic dermatitis. The process includes the step of determining, insera of the patient, the presence of antibodies against nuclear antigenssuch as transcription co-activator p75, wherein the presence of suchantibodies indicates atopic dermatitis. The screening process can beused to detect atopic dermatitis in patients suffering from otherconditions such as asthma or interstitial cystitis.

[0005] In accordance with the process, sera is obtained from the patientand contacted with the nuclear antigen (e.g., nuclear transcriptionco-activator p75). The resulting mixture is maintained for a period oftime sufficient for formation of an immune complex between antibodies inthe sera and the antigen. The antibodies can be of the IgG or IgE classof immunoglobulins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006]FIG. 1 (shown in two panels designated 1A and 1B) shows cloningand sequence date for DFS70. Panel 1A is a schematic representation ofoverlapping cDNAs. Clone 6.1 was obtained by immunoscreening a T24 cDNAlibrary with a human anti-DFS serum and clone 52 by 5′ RACE. Thecombined cDNA represented 3117 nucleotides. Panel 1B shows thenucleotide and deduced amino acid sequences of DFS-70. Sequencing ofboth DNA strands was performed with custom synthetic oligonucleotideprimers. The open reading frame starts at nucleotide 54 and ends atnucleotide 1645. The methionine start sequence and the polyadenylationsignals AATAAA/ATTAAA are underlined. The combined nucleotide and aminoacid sequences for the original clone 6.1 starting at nucleotide 593 andending at nucleotide 3117 was submitted to GenBank under accessionnumber U943319 in March 1997. The differences in nucleotides betweenDFS70 and p75 (residues single underlined) are at position 726 a→t(Glu→Gly) and 1314 a→t (Tyr→Phe) while there were no differences betweenDFS70 and LEDGE (lens epithelium derived growth factor) at thesepositions. DFS70 differed from both p75 and LEDGF (residues doubleunderlined) at position 188 g→t (Val→Phe), 244 g→a (no aa change) and347 a→g (Thr→Ala).

DETAILED DESCRIPTION OF THE INVENTION

[0007] The present invention provides a process of screening patientsfor atopic dermatitis. The process includes the step of determining, insera of the patient, the presence of antibodies against nuclear antigenssuch as transcription co-activator p75, wherein the presence of suchantibodies indicates atopic dermatitis. The screening process can beused to detect atopic dermatitis in patients suffering from otherconditions such as asthma or interstitial cystitis.

[0008] The identification of an antigen-antibody in AD was initiated byan observation in IC patients of an autoantibody against a nuclearantigen distributed as dense fine speckles in the necleoplasm of cellsin interphase and increased localization in condensed chromosomes inmitosis. In addition, in immunoblotting using tissue culture extract asthe antigen source, sera of such patients reacted with a 70 kilodalton(kDa) protein. This antigen was called DFS-70, incorporating theimmunolocalization of the antigen (dense fine speckles) and theestimated molecular size of the antigen in polyacrylamide-SDS gels (Ochset al., J Urol 1994; 151:587-592). The antibody was present in higherfrequency in AD patients than in IC patients. This antigen is a nuclearprotein, which is identical to a nuclear transcription co-activatorcalled p75. The antibody is also present in other conditions and it islikely that the underlying common feature is the presence of AD inpatients with asthma, interstitial cystitis and other disease states.

[0009] In accordance with the process of this invention, sera isobtained from a patient suspected on having AD. The sera is contactedwith the nuclear antigen (e.g., nuclear transcription co-activator p75).The resulting mixture is maintained for a period of time sufficient forformation of an immune complex between antibodies in the sera and theantigen. The antibodies can be of the IgG or IgE class ofimmunoglobulins. Means for collecting sera from patients are well knownin the art. Times and conditions that allow for immune complex formationare also well known in the art. Determination of immune complexformation can be accomplished any means known in the art. A specificexample of such determination is set forth hereinbelow.

[0010] The following teaching discloses a particular embodiment of thisinvention. A skilled artisan will readily recognize that otherembodiments can be used. Thus, the particular teachings set forth beloware not limiting of the specification and claims in any way.

[0011] Patients and Antibody Controls

[0012] Sixty-four patients with atopic dermatitis, from the Departmentof Dermatology, Nagoya University School of Medicine, Nagoya, JAPAN,were enrolled in our study. Diagnostic criteria for AD were thosedescribed by Hanifin and Rajka (Hanifin et al., Acta Dermatovener(Stockholm) Suppl 1980; 92:44-47). Chart records were reviewed for age,sex, duration of disease, eosinophil numbers, IgE levels, presence ofrespiratory atopy (bronchial asthma, allergic rhinitis), and degree offacial dermatitis. Patients with interstitial cystitis were diagnosedusing established criteria (Gillenwater et al., J Urol 1988;140:203-206) and were part of a large study on clinical features andsero-epidemiology reported previously (Koziol et al., J Urol 1993;149:465-469; Ochs et al., Interstitial Cystitis 1997, pp.47-52).

[0013] Patients with asthma were recruited from the Division of Allergyand Immunology, Scripps Clinic, La Jolla and fulfilled the AmericanThoracic Society criteria for the diagnosis of asthma. Psoriasispatients without accompanying atopic dermatitis came from a clinicalpractice. Patients with chronic fatigue syndrome were reported andcharacterized in a previous study (Konstantinov et al., J Clin Invest1996; 98:1888-1896). Normal human sera and sera from patients withsystemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome,and scleroderma were obtained from the serum banka of the W. M. KeckAutoimmune Disease Center, Scripps Research Institute, La Jolla, Calif.

[0014] Immunofluorescence

[0015] Sera from patients and controls were diluted {fraction (1/100)}in PBS and examined by indirect immunofluorescence for the presence ofautoantibodies to nuclear and cytoplasmic antigens on commerciallyprepared HEp-2 cell substrate slides (Bion, Park Ridge, Ill.) withanti-human IgG coupled to FITC (Caltag, San Francisco, Calif.) as thesecondary detecting reagent. IgG subclass identification ofautoantibodies was performed by indirect immunofluorescence oncommercially prepared HEp-2 cell substrate slides as described aboveusing affinity-purified FITC-conjugated sheep anti-human IgG1, IgG2,IgG3, and IgG4 (The Binding Site, Birmingham, England) as secondaryantibodies.

[0016] Immunoblotting

[0017] Human MOLT-4 cells (a T cell line) were obtained from theAmerican Type Culture Collection (Rockville, Md.) and grown insuspension culture in RPMI-1640 supplemented with 10% fetal bovineserum, 2 mM L-glutamine and 10 μg/ml gentamicin sulfate. Whole-cellextracts were prepared from MOLT-4 cells by pelleting suspended cells inmedia, rinsing in PBS, and resuspending the cell pellet in an equalvolume of 2× gel electrophoresis sample buffer containing a cocktail ofprotease inhibitors (catalog #1697498, Boehringer Manmheim,Indianapolis, Ind.). The cell extract was then sonicated on ice for 4-5bursts of 10 sec. each until the cells were dispersed. After sonication,the extract was then passed through progressively finer gauge needles,from #18 to #27, to shear the DNA and then the extract was boiled for 5min., centrifuged to remove insoluble material, and aliquots stored at−70° C. This form of cell extract has been used extensively in manyprevious studies (Ochs et al., Interstitial Cystitis 1997, pp.47-52;Konstantinov et al., J Clin Invest 1996; 98:1888-1896) as a source ofautoantigens for detection of serum autoantibodies against ubiquitouscellular antigens.

[0018] Western blotting was performed essentially as described by Chanand Pollard (Chan et al., Manual of Clinical Laboratory Immunology 1992,pp.755-762). Serum was diluted {fraction (1/100)} and detection ofimmunoreactive bands was performed by chemiluminescence usingperoxidase-conjugated goat anti-human IgG (Caltag, Burlingame, Calif.)and the ECL chemiluminescence kit (Amersham, Arlington Heights, Ill.)according to the manufacturer's directions, followed by autoradiography.For blotting of recombinant DFS70, the detection procedure wasidentical.

[0019] For detection of specific IgE autoantibodies to DFS70, serumsamples were diluted {fraction (1/10)} according to Valenta et al.(Valenta et al., J Invest Dermatol 1996; 107:203-208) in order to detectserum IgE levels that are on average 100,000 times lower in amountcompared to levels of IgG. IgE antibody was detected usingperoxidase-conjugated goat IgG antibody to human IgE epsilon heavy chain(Cappel, Durham, N.C.) as the secondary detecting reagent, followed bychemiluminescence and autoradiography as described above for thedetection of IgG. Normal control sera were included in everydetermination in order to exclude spurious findings in tlis procedure.

[0020] cDNA Cloning

[0021] DFS70 was originally described using autoantibodies from patientswith interstitial cystitis (Ochs et al., J Urol 1994; 151:587-592) andwas given the designation because of a characteristicimmunohistochemical staining pattern on HEp-2 (human epithelial Line)cells consisting of dense fine specldes distributed in the nucleoplasmin interphase cells and with accentuated generalized staining ofcondensed chromosomes in mitotic cells. These sera also showedreactivity with a 70 kDa protein in MOLT-4 extracts in Western blotting.One of these sera, which was high in antibody titer and available inlarger quantity, was used to isolate a partial cDNA sequence asindicated below.

[0022] The method for primary screening of a T24 (bladder carcinoma cellline) cDNA expression library was reported previously (Ochs et al.,Molec Biol Cell 1996; 7:1015-1024). Briefly, serum from patient 90-49was diluted {fraction (1/100)} and preabsorbed with wild-type λ ZapExpress phage without inserts. The preabsorbed serum was then used toscreen approximately 300,000 recombinant plaques from a cDNA expressionlibrary constructed from T24, a bladder epithelial cell line (ATCC,Rockville, Md.), using ¹²⁵I-staph protein A according to the method ofYoung and Davis (Young et al., Proc Natl Acad Sci (USA) 1983;80:1194-1198). All screenings were carried out on duplicate filters andtwo double-positive clones were obtained. After multiple rescreening,clone DFS6.1 was amplified, purified, and used for sequence analysis.Clone DFS6.1 was a partial cDNA and in order to isolate full-length cDNAmultiple overlapping 5′ clones were obtained from human placenta cDNAsusing a modified 5′-RACE method previously described (Lung et al.,Trends Genet. 1996; 12:389-391). Clone DFS6.1 was reported in anabstract (Ochs et al., Molec Biol Cell 1995; 6:75a Suppl.) and thenucleotide sequence was submitted to Gen Bank under accession No. U94319in March 1997.

[0023] Sequence and Protein Anaylses

[0024] cDNA inserts were analyzed by restriction mapping and sequencing.Nucleotide sequence was determined by dye primer cycle sequencing usinga Model 373A DNA sequencer from Applied Biosystems (ABI, Foster City,Calif.). Oligonucleotide primers were synthesized with a Model 394 DNAsynthesizer (ABI). DNA and protein sequences were analyzed by theGenetics Computer Group (GCG) Sequence Analysis Software Package version7.2 for UNIX computers (Deveraux et al., Nucleic Acids Res 1984;12:387-395). Alignment of protein sequences was achieved with the GAPprogram that employed the algorithm of Needleman and Wunsch. Molecularmass was calculated using the GCG program PEPTIDESORT.

[0025] Purification of Recombinant DFS70

[0026] For expression and purification of recombinant protein, theEcoR1-EcoR1 fragment of clone DFS6.1 and the fall-length cDNA weresubcloned into pET 28 vector (Novagen, Madison, Wis.). The full-lengthcDNA was obtained by RT-PCR using T24 cell mRNA and primerscorresponding to sequences flanking the methionine start codon and 3′stop codon. The sense and antisense primers were5′-GCAGAATTCGATACATGACTCGCGATTTC-3′ and5′-AGTGAAGCTTATATTCCAGGTATGTCAACCTACGTT-3′ and the sequencesincorporated to facilitate subdloning were EcoRI and HindIII restrictionsites. The pET28 vector has N-terminal fusion tags comprising T7 and 6×histidine. The T7 fusion tag allows for easy detection of recombinantprotein with T7 antibodies and the 6× histidines allow for proteinpurification due to the high affinity of histidine to nickel. E. colistrain BL21-DE3 containing the pET 28a vector with the DFS6.1 insert wasgrown in LB media containing kanamycin until OD₆₀₀=0.8. Then, 5 mM IPTGwas added to induce recombinant protein expression for 6 hrs. at 37° C.Cells were harvested by centrifugation and broken open by cycles offreezing and thawing in a buffer solution containing 6M guanidinehydrochloride according to the manufacturer's directions (Qiagen,Chatsworth, Calif.). This solution was then passed through aNi-NTA-Agarose resin (Qiagen, catalog #30210) column where therecombinant protein is bound at elevated pH (pH 8.0) by the affinity ofnickel for the 6× His portion, unbound material washed out, and therecombinant protein eluted in fractions at a pH of 6.0. Each fractionwas checked by Western blotting for the presence and purity ofrecombinant protein.

[0027] Recombinant Proteins and Antibodies for TranscriptionCoactivators p52 and 75

[0028] The USA (upstream stimulatory activity) fraction of HeLa cellscontaining the transcription coactivator proteins p52 and p75 wasdescribed previously (Ge et al., Cell 1994; 78:513-523). Subsequentstudies resulted in the cDNA cloning and production of recombinantproteins for p52 and p75 and the production of rabbit polyconalantibodies (Ge et al., EMBO J. 1998; 17:6723-6729). These reportsappeared while these studies were in progress and it was noted that ourplasmid DFS 6.1 had complete identity with the corresponding region ofp75. Studies were therefore initiated with Ge and colleagues (NIH,Bethesda, Md.) and reagents exchanged to determine the reactivity ofautoantibodies to p75 and p52 as described below.

[0029] Immunoelectron Microscopy

[0030] Small pieces of mouse intestine were fixed for 1 hr. at 4° C.with 1% glutaraldehyde buffered in PBS. Pieces were then dehydrated in70% ethanol and embedded in LR White. After polymerization at 56° C. for2 days, sections were collected grids. For immunolabeling, individualgrids (with sections down) were incubated for 30-60 min. with a blocldngsolution consisting of 2% NGS/0.2% BSA made in Tris-buffered saline(TBS) containing 1.0% Tween-20 (0.02 M Tris, 0.15 M Na Cl, 1.0%Tween-20, pH 7.6). Grids were then transferred to drops consisting of a{fraction (1/100)} dilution of human anti-DFS70 made in TBS/Tween-20 for1 hr. at R. T. and then rinsed with TBS/Tween-20 10×1 min. each. Gridswere then blocked for 30-60 min. in TBS/Tween-20 and incubated for 1 hr.at R. T. in a {fraction (1/50)} dilution of goat anti-human IgG linkedto 10 nm colloidal gold (Amersham) diluted in TBS/Tween-20. Grids werethen rinsed in TBS/Tween-20 10×1 min. each, rinsed in PBS, fixed 30 min.at R. T. in 1% glutaraldehyde/PBS, rinsed in PBS 3×5 min., rinsed indistilled water 3×5 min, counterstained with uranyl acetate and leadcitrate, rinsed in distilled water, and blotted dry.

[0031] ANAs in Patients with Atopic Dermiatitis

[0032] Sixty-four AD patients, 30 males and 34 females, were analyzedfor IgG autoantibodies to nuclear antigens (abbreviated as antinuclearantibody [ANA]) by indirect immunofluorescence on commercial human HEp-2cell substrates and by Western blotting on whole-cell extracts of humanMOLT-4 cells (FIG. 1). AD patients ranged in age from 4-43 yrs. (averageof 24.4 yrs.) and the duration of disease ranged from 1-35 yrs. (average13.4 yrs.). Of these 64 AD patients, 26 (40.6%) had ANAs of one patternor another but only 18 (28%) produced a pattern of dense fine-specklesin interhase nucleoplasm and staining of chromosomes in mitotic cells.TABLE 1 Clinical and Laboratory Data on 18 Atopic Dermatitis Patientswith DFS70-Staining Pattern in Immnunobistochemistry AntinuclearAntibodies Patient Sex Age^(a) Dur.^(b) Eos^(c) IgE^(d) R.A.^(e)F.D.^(f) Pattern^(g) Titer^(h) Anti-DFS70^(i) 6 F 20 15 ND ND BA/AR yesDFS/chr 1280 pos 8 F 4 2 400 67 no yes DFS/chr >1280 pos 10 F 23 22 2891859 No Yes DFS/chr 320 pos 12 M 19 14 1463 13226 BA/AR yes DFS/chr/NBs640 pos 13 F 20 16 659 4224 BA/AR yes DFS/chr 640 pos 15 F 29 14 1440 65no yes DFS/chr/No >1280 pos 16 F 22 13 566 6900 no yes DFS/chr 320 neg28 M 23 23 629 5024 AR yes DFS/chr 320 pos 36 F 20 6 213 488 AR yesDFS/chr 1280 pos 38 F 15 7 743 2189 no yes DFS/chr 320 pos 39 F 20 201296 6270 AR yes DFS/chr 1280 pos 41 F 19 9 885 196 no yes DFS/chr/NBs640 pos 68 F 20 8 568 1579 no yes DFS/chr 640 pos 77 F 27 26 975 1891 nono DFS/chr ND neg 78 M 21 9 87 1714 no yes DFS/chr ND neg 81 M 26 10 20525 no no DFS/chr 320 pos 88 F 19 3 300 84 AR yes DFS/chr/NBs 640 pos 99F 32 27 152 272 AR yes DFS/chr 640 neg

[0033] The prototypic immunofluorescence staining pattern AD sera showsthat interphase cell nuclei display fine specldes distributed in thenucleoplasm and aggregation of the specldes in the region of condensedchromosomes hi mitotic cells. A prototypic IC serum showed a similarpattern. Thirteen of the 18 patients described in Table 1 were includedin the Western blot analysis depicting the initial studies, which showedthat the majority of patients with DFS type antibodies appeared to bereacting with an antigen of approximately 70 kDa contained in MOLT-4cell extracts. Lanes 5 to 22 consisted of 13 sera from patients in Table1 with the addition of 5 other AD patients who did not have thedistinctive DFS type of autoantibodies and other control positive andnegative sera in lanes 1 to 4. It was noted that some AD sera hadantibodies to antigens of other molecular sizes but the importantfinding was the occurrence of a common antigen-antibody system in themajority of sea with DFS-type antibodies. The last column of Table 1includes data on the 18 DFS sera with respect to antibodies torecombinant antigen which is described firrther below. It should benoted from Table 1 that there was no significant relationship betweenthe presence anti-DFS-70 antibody and parameters of age, duration ofdisease, eosinophils and IgE levels or with respiratory atopy but themajority of patients had facial dermatitis.

[0034] cDNA Cloning of DFS70: Sequence Identity to tite TranscriptionalCo-activator p75

[0035] To characterize DFS70 and to determine whether or not it was anovel protein recognized by the sera of both AD and IC patients, we usedthe more abundant serum from IC patient 90-49 to clone the partial cDNAof DFS70. We first isolated poly A(+) RNA from human T24 bladderepithelial cells and from this starting material a cDNA expressionlibrary was constructed, screened with serum, and the positive clonesobtained were subeloned and purified to homogeneity. Clone DFS6.1,containing a 2 kb insert, was then cloned and sequenced (FIG. 1A).Analysis of this sequence showed an open reading frarne (ORF) ofapproximately 1 kb, coding for 351 amino acids with a predictedmolecular weight of 40 kDa (FIG. 1B). A search of the gene sequencedatabase at that time indicated no identity with previously reportedsequences. The sequence of cDNA clone DFS6.1 from protein DFS70 wasdeposited in GenBank under accession #U94319 (Ochs et al., Molec BiolCell 1995; 6.75a Suppl.) in 1997.

[0036] An overlapping clone #52 was obtained from human placental cDNAsusing 5′RACE (FIG. 1A). Independent confirmation was provided fromRT-PCR using primers upstream of the methionine start and translationstop codons. The amplified cDNA was subdloned into pET28 for sequencinganalysis as described in Methods. The complete cDNA and deduced aminoacid sequences are shown in FIG. 1B. GenBank search at this time showedthat DFS70 was identical to transcription coactivator p75 (Ge et al.,EMBO J 1998; 17:6723-6729) and lens epithelium-derived growth factor(LEDGF) (Singh et al., Invest Ophthalmol Vis Sci 1998; 39:3590). The twoto four amino acid differences among these proteins (see FIG. 1B) mayreflect errors in reverse transcriptase function during the cDNAsynthesis step for establishing the cDNA expression libraries sincethese are single nucleotide differences. It should be noted thatdifferent cell lines, T24 in this report, HeLa (Ge et al., EMBO J. 1998;17:6723-6729) and lens epithelial cell (Singh et al., Invest OphthalmolVis Sci 1998; 39:3590 (Abst)) cDNA libraries were used for isolating thedifferent cDNA clones.

[0037] Western Blotting: A Shiared Autoantibody/Autoantigen System in ADand IC Patients

[0038] For a higher level of protein expression and to purify therecombinant protein away from contaminating bacterial proteins thatmight interfere in Western blotting, the full-length cDNA clone of DFS70was excised and cloned into pET vector 28a (pET-DFS) that has a 5′ T7fusion protein tag and a 6× histidine tag. Recombinant DFS70 proteinexpressed from clone pET-DFS was bound and specifically eluted from anickel affinity column and was detected with either T7 antibody or theserum from IC patient 90-49 used to originally isolate clone 6.1 ofDFS70. Purified recombinant DFS70 was then used to confirm the presenceof IgG antibodies originally detected using MOLT-4 cell extracts.Patients that were positive in Western blotting against a 70 kDa proteincontained in MOLT-4 cells and were also positive against recombinantDFS70. All 64 AD sera were subsequently examined in Western blottingagainst recombinant DFS70 and 19 (29.6%) were positive (see Table 2).TABLE 2 Patient Groups Reactive with Recombinant DFS70 by WesternBlotting Patients Reactivity Normal Controls 0/39 (0%) Atopic Dermatitis19/64 (29.6%) Asthma 8/50 (16%) Interstitial Cystitis 9/103 (8.7%)Psoriasis 1/22 (4.5%) Chronic Fatigue Syndrome 2/60 (3.3%) SystemicLupus Erythematosus 0/36 (0%) Rheumatoid Arthritis 0/30 (0%) Sjögren'sSyndrome 2/29 (6.9%) Scleroderma 1/40 (2.5%)

[0039] In summary, of 18 sera showing putative DFS staining patern byimmunohistochemistry, 14 were positive in Western blotting againstrecombinant DFS70 and of the 46 AD sera without this pattern inimmunohistochemistry, 5 were positive in Western blotting.

[0040] The IgG subclass was determined for 16 AD sera that were reactivefor DFS70. All antibodies were of the IgG1 subclass and none were IgG4.Two sera also contained antibodies of the IgG2 subclass and 3 otherscontained antibodies of the IgG3 subclass.

[0041] Detection of IgE Autoantibodies to DFS70

[0042] Since blood levels of IgE are approximately 100,000 times lessthan those for IgG, we performed IgE Western blotting at serum dilutionof {fraction (1/10)} compared to normal dilutions of {fraction (1/100)}for IgG, and the signals obtained also necessitated much longerautoradiographic exposure times (20 minutes for IgE antibody versus 5seconds for IgG antibody). In both IC and Ad patients we could detectspecific IgE autoantibodies to DFS70 by Western blotting. It was notedthat in general, there was a positive relationship between signals forIgG and for IgE antibodies in that a strong signal for IgG antibodycorrelated with a strong signal for IgE antibody.

[0043] Other Patient Groups Reactive with Recombinant DFS70

[0044] Table 2 summarizes the results of Western blotting againstrecombinant DFS 70 protein in various groups of patients and normalcontrols. None of the normal healthy controls were positive for IgGautoantibodies reactive with recombinant DFS70 compared to 30% of ADpatients. This result compares with a positivity of 16% for asthmapatients, 8.7% for IC, 4.5% for psoriasis patients, 3.3% for patientsdiagnosed with chronic fatigue syndrome, 0% for lupus patients, 0% forrheumatoid arthritis patients, 6.9% for Sjogren's patients, and 2.5% forpatients with scleroderma.

[0045] Human Autoantibodies to DFS70 Bind Transcription Coactivator p75,but not p52

[0046] During the course of the studies described above, a report by Geet al. (Ge et al., EMBO J. 1998; 17:6723-6729) described the isolationof cDNAs encoding transcription coactivators p52 and p75, which theseauthors showed were required for transcriptional activation in humancell-free systems containing RNA polymerase It and general initiationfactors. p52 is a protein of 333 aa and is a splice variant of a genethat also encodes a protein p75 of 530 aa with the additional aminoacids of p75 located in the C-terminal region. Our initial DFS6.1partial cDNA clone encoded for the C-terminal 351 aa of p75 and it wasof interest to determine how AD sera reacted with the splice variantsp75 and p52. Five human sera, four from patients with atopic dermatitis(LB, KK,CK, WS) and an autoirnmune serum containing autoantibodies toother cellular antigens were coded and analyzed for reactivity in the Gelaboratory with the upstream stimulatory activity (USA) fraction of HeLacells which contained both p52 and p75. All the atopic dermatitis serareacted with p75 but not with p52 whereas the irrelevant autoimmuneserum AF reacted with USA fraction to give two unrelated bands ofunknown identity. Rabbit antibodies raised against p52 and p75 fractionswere used as positive controls: anti-p52 reacted with both p52 and p75whereas anti-p75 reacted with p75 alone. This could be expected sinceanti-p52 was raised with full-length p52 as immunogen whereas anti-p75was raised with the unique C-terminal region of p75. The human sera werefuirther analyzed in Western blotting with recombinant p75 and p52, withthe results confirming the observations made with the USA fraction.

[0047] Rabbit Antibodies to Recombinant p75 and p52 Bind to RecombinantDFS70 and Display DFS Localization

[0048] It was demonstrated that rabbit antibodies raised againstrecombinant transcription factors p75 and p52 were both able to bind torecombinant full-length DFS70. When the rabbit antibodies were used inimmunohistochemistry, they displayed identical patterns ofimmunolocalization as compared to human anti-DFS70. A pattern of densefine speckles in interphase nuclei and generalized staining of condensedchromosomes in mitotic nuclei was observed.

[0049] Localization of DFS70 at the Ultrastructural Level

[0050] A high titered human anti-DFS70 was selected for immunoelectronmicroscopy and the targets were small pieces of mouse intestine fixed at4° C. with 1% glutaraldehyde as described in Materials and Methods.Mouse intestine was used as the substrate because p75 has been shown tobe ubiquitously expressed at the mRNA level in many different tissuesincluding small intestine (Ge et al., EMBO J. 1998; 17:6723-6729) andnumerous studies have shown that human autoantibodies react withcellular antigens which are highly conserved between mouse and man (Tan,Cell 1991; 67:841-842). Immunoelectron microscopy demonstrated thatDFS70/p75 was localized in interphase chromatin and appeared to beconcentrated over areas of condensed chromatin in the periphery of thenucleus and also in perinucleolar chromatin. The nucleolus and theinterchromatin regions contained relatively few colloidal goldparticles.

[0051] As shown in Table 1, 18 AD patients had what appeared to be acharacteristic DFS staining pattern by immunohistochemistry, but four ofthese sera (16,77,78,99) were negative in Western blotting againstrecombinant DFS70. It is possible that if these four sera did haveantibodies to DFS70/p75 transcription coactivator, the epitope(s)recognized by the four sera might not be primary sequence regions of theprotein but are conformation-dependent epitopes which are lost duringdenaturation in the polyacrylamide gel electrophoresis-Wester blottingprocedure. Alternatively, immunohistochemistry might be more sensitivethan immunoblotting for this antigen-antibody system. It is also ofinterest five AD patients which did not display the prototypic DFS70staining pattern were found to have antibody to recombinant DFS70 inWestern blotting.

[0052] The DFS70/p75 autoantibody/autoantigen system showed the highestprevalence in three disease conditions, i.e., atopic dermatitis (30%),asthma (16%) and interstitial cystitis (9%). It has been well documentedthat AD is frequently associated with bronchial asthma and that childrenwith AD are more likely to develop asthma in later life than non-ADchildren (Cooper, J Invest Dermatol 1994; 102:128-137; Beltrani, JAllergy Clin Immunol 1999; 104:587-598). In addition to the fact thatANAs are present in higher than normal frequency in IC patients, thereare several reports that allergic disorders are a major symptomcomplaint in IC. In a recent study on 374 patients, food allergies werereported in 25.1% and hay fever in 24.9% (Kozial et al., J Urol 1993;149:465-469). Earlier studies have also pointed out the commonoccurrence of allergic disorders in IC and the role played by mast cells(Messing, et al. Urology 12:381-392; Hanno et al., J Urol 1990;143:278-281). None of these studies reported on the frequency of atopicdermatitis and it might be possible that in IC, some of the patientswith allergic diathesis might also have AD and that this could be acommon thread running through the three different disease condition.

[0053] It is of interest that the putative function of the DFS70/p75antigen has been reported to be a transcription co-activator (Ge et al.,EMBO J. 1998; 17:6723-6729). There are two isoforms which are splicedproducts of the same gene and encode for p75 as well as a truncated p52kilodalton protein which lacks the C-termninal region of p75. In studiesshowing that both these proteins functioned as transcriptionco-activators in an in vitro transcription system dependent on RNApolymerase II and general transcription factors, the shorter isoform p52was more active for most activation domains compared to p75. Humanautoantibodies recognized exclusively the p75 isoform, indicating thatthe epitope was located in the C-terminal region of p75 which is absentin p52. We and others have advanced the concept that spontaneouslyoccurring autoantibodies are antigen-driven (Tan, The Immunologist 1999;7:85-92; Radic et al., Ann Rev Immunol 1994; 12:487-520) and if this isthe case for the autoimmune response to DFS70, the immunogen which isdriving the immune reactin is the larger isoform, p75. At the presenttime, the significance of this finding is unknown, but although p75 is aless active transcription co-activator, it is possible that itsinvolvement in transcription might be related to certain specificactivation domains which were not used in the reported studies (Ge etal., EMBO J. 1998; 17:6723-6729). In this respect, future studies couldaddress the question whether p75 might enhance transcription activityfor certain genes predisposing to the development of atopy (Forrest etal., J Allergy Clin Immunol 1999; 104:1066-1070).

[0054] Our studies using autoantibodies from patients with AD and IC,and antibodies from rabbits immunized with p75 show that DFS70/p75 islocalized in the nucleoplasm by immunohistochemistry. This is consistentwith the function of p75 as a transcription co-activator associated withRNA pol II. Irmunoelectron microscopy studies also show that DFS70 islocalized in the nucleus and preferentially in heterochromatin areas.This is in contrast to the reports by Singh et al. (Singh et al., InvestOphthalmol Vis Sci 1999; 40:1444-1451) on the same protein which theycall lens epithelium derived growth factor (LEDGF) which was initiallyreported to be a cytoplasmic protein (Singh et al., Invest OphthalmolVis Sci 1998; 39:3590 (Abst)) and subsequently reported to be present inthe cytoplasm of lens epithelial cells at 4°, 15° and 28° and in thenucleus and nucleolus at 37° (Singh et al., Invest Ophthalmol Vis Sci1999; 40:1444-1451). Its localization in the cytoplasm at temperatureslower than 37° is non-physiological and the significance of this isunknown. These authors identified LEDGF and isolated a cDNA encodingthis protein with the serum of a patient with age-related cataract. Someof the sera from patients with age-related cataract appeared to containantibodies to LEDGF. The interesting feature is that cataracts and otherocular complications have been reported in certain patients with AD(Nakano et al., J Jpn Ophthalmol Soc 1997; 101:64-68; Fagerhold et al.,Graefes Arch Clin Ophthalmol. 1984; 221:149-152) and in one report, lensopacities were noted in 23.8% of 133 eyes examined (Nakano et al., J JpnOphthalmol Soc 1997; 101:64-68).

[0055] We have proposed the notion that spontaneously occurringautoantibodies might be regarded as messengers from the immune systemthat report abnormal events which involve cellular componentsparticipating in disease-related mechanisms and that these cellularcomponents might be the antigens driving the immune response (Tan, TheImmunologist 1999; 7:85-92). The autoantibodies themselves might not beplaying a pathogenic role, but can be used to identify the cellularcomponents participating in such disease processes. However, thequestion which needs to be addressed is whether the autoimune process inAD involving DFS70/p75 is playing any role in pathogenesis. The currentstudy does not address the question of the cell mediated immune systemsince cellular infiltrates consisting of increased numbers oflymphocytes, monocytes, macrophages and mast cells are present in atopiclesions (Cooper, J Invest Dermatol 1994; 102:128-137; Leung, J AllergyClin Immunol 1999; 104:S99-S108). An important study would beexamination of slin biopsies from AD lesions to determine whether theremight be abnormal expression of DFS70/p75 mRNA and protein. Such studiescould not be done since the present work was carried out only with serumspecimens and further studies along these lines are needed. We did findIgE autoantibodies to DFS70/p75 in both AD and IC patients and thisfinding lends some support to the possible pathogenic significance ofthe present findings in view of several studies showing prominence ofIgE positive Langerhans cells in AD lesions (Leung, J Allergy ClinImmunol 1999; 104:S99-S108).

What is claimed is:
 1. A process of screening a patient for atopicdermatitis comprising determining in sera of the patient the presence ofantibodies against nuclear transcription co-activator p75, wherein thepresence of such antibodies indicates atopic dermatitis.
 2. The processof claim 1 wherein the patient is asthmatic.
 3. The process of claim 1wherein the patient has interstitial cystitis.
 4. The process of claim 1wherein sera from the patient is contacted with and maintained for aperiod of time sufficient for formation of an immune complex betweenantibodies in the sera and the
 5. The process of claim 1 wherein theantibodies are IgG molecules.
 6. The process of claim 1 wherein theantibodies are IgE molecules.